NAD⁺ exhaustion–induced Sirt3 dysfunction is responsible for CPT2 K79 acetylation and acylcarnitine accumulation. (A) NAD⁺ levels of stored human platelets were detected. (B) The aggregation, apoptosis, MMP, and mtROS levels were measured in human platelets stored for 6 days with 0.5 mM NMN, 0.5 mM nicotinamide riboside (NR), 2.5 µM P7C3, or 10 µM SBI797812. n = 3. (C) Apoptosis, mtROS levels, and MMP of stored WT and Sirt3^−/− platelets were detected. n = 3. (D) Subcellular location of the proteins in which acetylation levels were changed (2-day Sirt3^−/− vs 2-day WT, fold ≥1.3). (E) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment of the proteins in panel D. (F) The acetylation sites of the enzymes that were involved in fatty acid metabolism are listed. (G) The levels of CPT2 K79 acetylation were detected in WT and Sirt3^−/− platelets stored for 0 day and 1 day by using western blot analysis. CPT2 was used as a loading control. (H) The levels of C16:0 and C18:0 acylcarnitines were evaluated in WT and Sirt3^−/− platelets stored for 0 day or 1 day, respectively. n = 3. *P < .0332, **P < .0021, ***P < .0002, ****P < .0001.